Hyphal-Mediated Nitrogen translocation is Optimized at Intermediate N Levels in Pinus massoniana from 15N Tracing

Aims Common mycorrhizal networks (CMNs) serve as essential pathways for belowground resource exchange, with a key function being hyphal-mediated nitrogen translocation across plant hosts. This study specifically investigates how CMNs, established by mixed mycorrhizal communities, facilitate interplant nitrogen translocation and regulate nitrogen allocation strategies. By elucidating this mechanism, our work provides critical insights for the restoration of degraded forest ecosystems. Methods This study developed a four-cell grid seedling apparatus (A-D chambers) to physically isolate root contact while preserving mycelial network connectivity. A sterile soil medium was used to maintain hyphal viability and prevent inter-chamber nutrient diffusion. A controlled experiment was conducted using Pinus massoniana seedlings inoculated with mixed mycorrhizal strains (Sm, comprised Pisolithus orientalis (Po), Scleroderma citrinum (Sc), and Suillus luteus (Sl), isolated and purified from P.massoniana rhizosphere soil samples.) versus non-inoculated controls (CK). Different nitrogen treatments were carried out through (¹⁵NH₄)₂SO₄ solution isotope labeling (0 g/L (N0), 2 g/L (N2), 4 g/L (N4), and 6 g/L (N6)), we quantitatively assessed hyphal-mediated nitrogen translocation through CMNs across a 30-µm mesh barrier. Results CMNs established direct hyphal connections between donor and receiver seedlings, facilitating nitrogen translocation via hyphal pathways. The translocation pattern, which peaked at intermediate N levels, indicates a regulated interplant nitrogen redistribution mediated by the fungal network, rather than a mere enhancement of direct uptake by the receiver's mycorrhizae. Sm-inoculated seedlings exhibited a 2.62-fold increase in root biomass (N6) and 50.59% leaf nitrogen allocation (N4), significantly surpassing CK. The Sm-N4 treatment demonstrated optimal performance, enhancing photosynthetic capacity via prioritized nitrogen allocation to leaves. Notably, ¹⁵ N transfer ratios directly correlated with receiver seedling biomass and nutrient uptake, highlighting CMNs as a functional conduit for resource redistribution. Conclusions Our study shows that plants rely more on mycorrhizal fungi for nitrogen (N) when soil N levels drop. This dependence peaks at a critical threshold (N4), where fungal N translocation is most efficient. This supports the carbon-for-nitrogen trade model, where plants exchange carbon with fungi to acquire nitrogen, particularly for leaf development under N scarcity. Identifying N4 as the optimal level helps guide mycorrhizal use in reforesting N-poor areas.